T cell receptor sequence analysis indicates that myelin specific T cells are expanded in multiple sclerosis (MS) patients, but adequate techniques for the enumerization of antigen-specific T cells in relationship to the disease process are not available. We hypothesize that the frequency of T cells specific for myelin-derived peptides has been greatly underestimated by limiting dilution techniques. Analysis of antigen-specific CD4+ T cells with sensitive techniques will have important implications for understanding the pathogenesis of MS and for developing adequate tools for immune monitoring. The analysis of CD8+ T cells in viral infections has been revolutionized by the creation of tetrameric forms of MHC class I/peptide complexes. Massive expansion of antigen-specific CD8+ T cells has been documented in both acute and chronic viral infections. The introduction of this technology led to the realization that the frequency of antigen specific CD8+ T cells in viral infections had been greatly underestimated. The generation of tetrameric forms of human MHC class II molecules may have a major impact on the investigation of human autoimmune disease. An expression system for soluble HLA-DR2 was previously developed for crystallization of HLA-DR2 with a bound peptide form human myelin basic protein. Based on this expression system, tetramers of HLA-DR2/peptide complexes have been generated with fluorescently labeled streptavidin. These molecules will be used to determine the frequency, activation state and cytokine profile of antigen specific T cells in the cerebrospinal fluid and blood of MS patients with the HLA-DR2 haplotype. The tetramer approach will also be used to generate multivalent TCRs using cDNAs derived from myelin specific T cell clones. MS plaque tissue will be stained with these multivalent TCRs to examine surface expression of HLA-DR2-bound myelin peptides by antigen presenting cells in the target organ.